The broad, long-term objective of this proposal is to understand the molecular signaling events during AIDS neuropathogenesis and blood-brain barrier (BBS) disruption. Encephalitis, characterized by perivascular cuffing of infected monocyte-derived macrophages (MDMs), is associated with BBB disruption. It is not known if the MDM infiltration results in BBB disruption, or is facilitated by disruption. We propose to infect macaques with SIVmac251, and increase the incidence of encephalitis by depleting CD8 cells using a three-stage depletion strategy routinely used in rhesus macaques. We will then determine the mechanisms of interendothelial junction disruption triggered during AIDS neuropathogenesis. A key regulator of endothelial function is focal adhesion kinase (FAK). The central hypothesis to be explored is that 1) focal adhesion kinase activation is a key mechanism by which BBB breakdown occurs during neuroAIDS pathogenesis, and 2) that this activation requires the presence of productively infected monocytes. The following specific aims are proposed, using the rhesus macaque: SA-1: Determine if a productively- infected monocyte/ macrophage crossing the BBB induces activation of FAK and disruption of tight junctions. We will model neuroinvasion using an in vitro model of the BBB to determine if productively-infected cells are required to have physically crossed the BBB for this breakdown. SA2: Determine if phosphorylation of FAK is a requirement for BBB disruption and development of encephalitis. SA-2a: Experiments in vivo. Expression of FAK and tight junction proteins will be assessed using confocal microscopy. We will also examine thick section confocal microscopy of brains of macaques infected with SIVmac251 to determine association of productively infected macrophages with areas of BBB disruption. BBB integrity will be monitored by ELISA for the oligodendrocyte protein 5100(3 to determine timing of lesion formation. SA-2b: Experiments ex vivo. SA-2b will assess the timing and mechanisms of BBB disruption by immunocytochemistry and protein expression/ activation. FAK is activated by phosphorylation of key tyrosine residues. We will immunoprecipitate using phosphotyrosine antibodies and immunoblot for FAK, followed by band densitometry. We expect to determine the timing and mechanisms of BBB breakdown during the development of AIDS from the in vivo data, with real time information coming from ex vivo and in vitro studies. This research is important because the era of HAART is resulting in an increased cumulative prevalence of AIDS-related CMS complications. If we can determine how BBB is breaking down in SIV infection then we can develop new strategies for prevention of HIVE and the associated peripheral neuropathies.